Alternative miRNA Biogenesis Pathways and the Interpretation of Core miRNA Pathway Mutants

Molecular Cell

Review Alternative miRNA Biogenesis Pathways and the Interpretation of Core miRNA Pathway Mutants Jr-Shiuan Yang1,2 and Eric C. Lai1,* 1Department

of Developmental Biology, Sloan-Kettering Institute, 1275 York Avenue, Box 252, New York, NY 10065, USA Biology Program, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.07.024 2Molecular

Since the establishment of a canonical animal microRNA biogenesis pathway driven by the RNase III enzymes Drosha and Dicer, an unexpected variety of alternative mechanisms that generate functional microRNAs have emerged. We review here the many Drosha-independent and Dicer-independent microRNA biogenesis strategies characterized over the past few years. Beyond reflecting the flexibility of small RNA machineries, the existence of noncanonical pathways has consequences for interpreting mutants in the core microRNA machinery. Such mutants are commonly used to assess the consequences of ‘‘total’’ microRNA loss, and indeed, they exhibit many overall phenotypic similarities. Nevertheless, ongoing studies reveal a growing number of settings in which alternative microRNA pathways contribute to distinct phenotypes among core microRNA biogenesis mutants.

Introduction MicroRNAs (miRNAs) are abundant
22 nucleotide (nt) regulatory RNAs, derived from endogenous short hairpin transcripts, that collectively play key roles in diverse developmental and physiological processes in most eukaryotes (Flynt and Lai, 2008). The general defining features of miRNA genes are cleavage of their precursor transcripts by one or more RNase III enzymes, and sorting of mature species into Argonaute proteins of the Ago subfamily (Axtell et al., 2011). As with other classes of Argonaute-bound small RNAs, miRNAs serve as antisense guides to identify regulatory targets (Czech and Hannon, 2011). Plant miRNAs frequently pair extensively with one or a few targets, and these interactions have reliably proven to mediate their key functions (Axtell et al., 2011). In contrast, animal miRNAs have propensity to recognize targets via
7 nt complements to their 50 ends (preferentially nucleotides 2–8, the miRNA ‘‘seed’’) (Bartel, 2009). Computational and experimental strategies provide evidence for hundreds to 1000 direct conserved targets for individual human miRNAs, such that a majority of human transcripts carry conserved binding sites for multiple miRNAs (Bartel, 2009). The broad nature of animal miRNA target networks has made it difficult to infer phenotypically relevant aspects of miRNA biology. Moreover, many individual miRNA mutants have subtle phenotypes, and relatively few miRNA knockouts have yet been reported in many species, including most vertebrates (Smibert and Lai, 2008). Instead, knockouts of core miRNA factors are commonly used as a proxy to assess the phenotypic effects of removing miRNAmediated regulation. Over a hundred studies have studied straight or conditional knockouts of mouse dicer (Bernstein et al., 2003; Harfe et al., 2005; Kanellopoulou et al., 2005; Yi et al., 2006), and they collectively show this enzyme to be required for normal development, differentiation, and/or physiology of most tissues. In some cases, dicer phenotypes can be 892 Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.

causally linked to removal of specific miRNAs. For example, conditional knockout of dicer in the B cell lineage blocks the transition from pro-B to pre-B cells, accompanied by global upregulation of many targets of the mir-17-92 cluster, including the propapoptotic miR-17-92 target Bim (Koralov et al., 2008). These phenotypes were shared by knockout of the mir-17-92 cluster, which is highly expressed in the B cell lineage (Ventura et al., 2008), suggesting that it is responsible for a substantial aspect of the dicer mutant phenotype. In zebrafish, a compelling illustration was the rescue of early embryogenesis in dicer mutants by injection of a single small RNA duplex for miR-430, the major early-expressed miRNA in this species (Giraldez et al., 2005). While it is generally reasonable to infer that phenotypes of mutants such as dicer are due to miRNA loss, functional connections to individual miRNAs are often correlative, especially in the intact animal. Phenotypes may stem from concomitant loss of multiple miRNAs, and there are technical challenges to re-expressing functional miRNAs in biogenesis mutants. Furthermore, the existence of alternative miRNA pathways raises the possibility that core biogenesis mutants maintain subclasses of active miRNAs, or conversely that their phenotypes do not simply reflect the removal of miRNAs. The goals of this review are thus twofold. First, we describe the diversity of alternative miRNA biogenesis mechanisms, which reflect evolutionary flexibility in the acceptance and routing of different sources of double-stranded RNA by RNase III enzymes and Ago proteins. Second, we discuss biological settings in which loss of different core miRNA machinery has distinct consequences for depletion of miRNAs and related regulatory RNAs, and how this may impact the interpretation of organismal phenotype. The extensive literature on plant miRNAs notwithstanding, we focus this review on animal systems, for which alternate miRNA pathways are more abundant and the biological roles of miRNAs less well understood.

Molecular Cell

Review A Drosha/DGCR8-dependent

B Drosha/DGCR8-independent

Dicer-dependent

C Drosha/DGCR8-dependent

Dicer-dependent

Dicer-independent 3’ tailed mirtron

mirtron

5’ tailed mirtron

mir-144

canonical miRNA

exon

pri-miRNA Pol II

Drosha AAAA...3’

5’

exon

exon

exon

mir-451

exon

exon

Splicing and debranching endo-shRNA (and tailed shRNA)

DGCR8

Drosha 5’

nuclease?

AAAA...3’

DGCR8

Exosome

5’-3’ trimming

3’-5’ trimming

UU

ppp

pre-miRNA

Ago2

Dicer

Slicing endo-siRNA substrates hpRNA

TEs, trans-NATs (pseudogenes)

Dicer Ago2

miRNA/ miRNA* duplex

Cleavage ?

cis-NATs

Ago1-4 RNase Z RNase Z

mature miRNA

Ago1-4

alternative termination?

Ago2

Ago1-4

(siRNAs)

(miRNAs)

5’

U U 3’

trimming, tailing?

Ago2

5’ U U 3’

Pol III

MHV tRNA:tandem hairpins

Figure 1. Canonical and Major Alternative miRNA Biogenesis Pathways in Animals (A) Canonical animal miRNAs are generated through consecutive cleavages of hairpin precursors by two RNase III enzymes. In the nucleus, the single stranddouble strand junction of the pri-miRNA hairpin is recognized by DGCR8, which positions the catalytic site of the RNase III enzyme Drosha. This cleavage generates an
55–70 pre-miRNA hairpin that is exported to the cytoplasm, where it is cleaved toward the terminal loop end by the RNase III enzyme Dicer. The miRNA/miRNA* duplexes are loaded into miRNA-class Argonaute effectors (in mammals, Ago1–4). One of the duplex strands is preferentially retained in Ago to form the functional RNA-induced silencing complex. (B) Many Drosha/DGCR8-independent pathways can generate pre-miRNA-like hairpins that serve as Dicer substrates. Mirtrons are short intronic hairpins that are excised by splicing and linearized by lariat debranching; tailed mirtrons require further resection by nucleases, e.g., 30 -50 resection of 30 -tailed mirtrons by the RNA exosome. RNA pol III-transcribed MHV68 tRNA-shRNA fusions are processed into pre-miRNA-like hairpins with defined 50 and 30 ends as a result of RNaseZ cleavage and pol III termination, respectively. Endo-shRNAs without lower stems for Drosha/DGCR8 processing may derive from pol III transcription or cleavage by as yet unknown endo- or exonucleases. These noncanonical miRNAs, like canonical miRNAs, are incorporated to Ago1–4. Endogenous substrates with extended dsRNA character, including hpRNAs, transposable elements (TEs), antisense pseudogenes and natural antisense transcripts (NATs), are directly cleaved by Dicer to generate siRNAs. These may potentially sort to all of the mammalian Agos, but presumably only those that load Ago2 can fulfill target slicing. (C) Pri-mir-451 is processed by Drosha/DGCR8, and the resulting
18 bp pre-mir-451 is directly incorporated to Ago2. The Slicer activity of Ago2 cleaves the 30 arm of pre-mir-451, giving rise to ac-pre-mir-451, which is further resected by an as yet unknown mechanism to generate mature miR-451.

Part I: Canonical and Alternative miRNA Biogenesis Pathways The Canonical miRNA Biogenesis Pathway A canonical pathway driven by RNase III enzymes generates the majority of animal miRNAs (Ghildiyal and Zamore, 2009) (Figure 1A). Primary miRNA (pri-miRNA) transcripts are typically products of RNA Polymerase II, and the hairpins are usually contained within noncoding RNAs or the introns of messenger RNAs (mRNAs); frequently, multiple pri-miRNA hairpins are encoded by an individual transcript. Their biogenesis begins with cleavage near the base of each pri-miRNA hairpin by the nuclear Drosha/ DGCR8 heterodimer. DGCR8 (known as Pasha in invertebrates) is a double-stranded RNA (dsRNA) binding protein that recognizes the proximal
10 bp of stem of the pri-miRNA hairpin, positioning the catalytic sites of the RNase III enzyme Drosha (Han et al., 2006). Cleavage releases a pre-miRNA hairpin that is typically
55–70 nt in length. The
2 nt 30 overhangs of pre-miRNA hairpins are recognized by Exportin-5 (Exp-5) and its partner Ran-GTP, enabling their nuclear export. In the cytoplasm, the Dicer RNase III enzyme

cleaves pre-miRNAs
2 helical turns into the hairpin, yielding
22 nt small RNA duplexes. One of the strands is usually preferentially incorporated into an effector Ago protein and guides it to targets (Czech and Hannon, 2011). Select miRNA:target pairs in animals exhibit extensive complementarity permitting their cleavage by Slicer-class Ago proteins (Karginov et al., 2010; Shin et al., 2010; Yekta et al., 2004). However, the bulk of miRNA targets lack sufficient pairing for slicing, and are instead repressed by deadenylation, mRNA degradation, and/or translational suppression (Fabian et al., 2010). While Dicer and Ago proteins are central to miRNA biogenesis in all species, certain homologs have distinct properties (Ghildiyal and Zamore, 2009). For example, C. elegans and vertebrates encode a single Dicer that generates both miRNAs and small interfering RNAs (siRNAs), but Drosophila encodes two Dicers, of which Dcr-1 is specialized for pre-miRNA cleavage and Dcr-2 is selective for siRNA biogenesis. Argonaute proteins also exhibit specialization. Drosophila has two Ago-class effectors, of which AGO1 is dominant for miRNAs and AGO2 for siRNAs; these are further specialized in that AGO2 is a more Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc. 893

Molecular Cell

Review effective Slicer than AGO1, and AGO2-resident species are modified by 20 O-methylation. All four vertebrate Ago-class effectors participate in miRNA-mediated regulation and carry similar miRNA contents; thus, they lack comparable sorting mechanisms that distinguish Drosophila AGO1 and AGO2 cargoes. However, only Ago2 among vertebrate Ago proteins has Slicer activity, implying that it has unique activities for small RNA biogenesis and/or function. The Mirtron Pathway Deep sequencing of D. melanogaster revealed short RNA duplexes mapped to short hairpin introns, termed ‘‘mirtrons,’’ where the mature small RNA termini coincided with splice acceptor and donor sites (Okamura et al., 2007; Ruby et al., 2007). This suggested that splicing might substitute for Drosha cleavage, and this indeed proved to be the case (Figure 1B). As with other introns, the splicing reaction generates a nonlinear intermediate that must be resolved by the lariat debranching enzyme before the hairpin structure can be adopted. At this step, mirtron products appear as pre-miRNA mimics and enter the canonical biogenesis pathway as Exp-5 and Dcr-1 substrates, yielding mature products that populate AGO1 and can regulate typical seed-matching targets. Mirtrons are prevalent in both D. melanogaster and C. elegans (Chung et al., 2011), perhaps exploiting the fact that their genomes contain an abundant class of short introns that overlaps the length of pre-miRNAs (Lim and Burge, 2001). In fact, one of the earliest annotated worm miRNA genes (mir-62) was later recognized as a mirtron (Ruby et al., 2007). Subsequently, mirtrons were recognized in diverse vertebrates (Babiarz et al., 2008; Berezikov et al., 2007; Glazov et al., 2008). Cloning of murine small RNAs from dgcr8 or drosha knockout cells verified near-complete loss of canonical miRNAs, but maintained expression of mirtron-derived miRNAs (Babiarz et al., 2008; Chong et al., 2010; Yi et al., 2009). Therefore, vertebrate mirtrons probably follow a similar maturation pathway as in invertebrates. Tailed Mirtrons: 50 versus 30 Tails With conventional mirtrons, both ends of the pre-miRNA are defined by splicing. However, in the atypical locus Drosophila mir-1017, only the 50 hairpin end matches the splice donor site, followed by an
100 nt unstructured tail before the splice acceptor site (Ruby et al., 2007). Conversely, there exist vertebrate introns where 30 hairpin ends coincide with splice acceptor sites, but are preceded by unstructured tails following their splice donor sites (Babiarz et al., 2008; Glazov et al., 2008). Presumably such ‘‘tailed mirtrons’’ are processed by splicing, but require additional biogenesis steps (Figure 1B). The biogenesis of Drosophila 30 -tailed mirtrons was recently reported to utilize the RNA exosome (Flynt et al., 2010), the major 30 -50 exoribonuclease in eukaryotes. In this pathway, the 30 tail of the spliced and debranched full-length intron is removed by the exosome to yield the pre-miRNA. In vitro assays showed that the exosome was inhibited by the hairpin structure, allowing for pre-mir-1017 release. As with conventional mirtrons, pre-mir1017 is then cleaved by Dcr-1 and loaded into AGO1 to function as a typical miRNA. The biogenesis of mammalian 50 -tailed mirtrons has not been elucidated, but their configuration suggests potential involvement of 50 -30 exoribonucleases, such as the XRN family. Thus far, 30 -tailed mirtrons have only been 894 Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.

described in Drosophila, while 50 -tailed mirtrons have only been annotated in vertebrates, suggesting adoption of distinct hybrid pathways for splicing-mediated miRNA biogenesis in different animal clades. Box H/ACA- and Box C/D snoRNA-Derived miRNAs Small nucleolar RNAs (snoRNAs) have analogies to miRNAs in that they are also abundant, deeply conserved short RNAs that serve as antisense guides. In their best-known roles, snoRNAs guide posttranscriptional modifications of ribosomal RNA (rRNA) and snRNA targets. The presence of submotifs permits snoRNAs to be categorized as C/D box or H/ACA box classes, which typically mediate 20 -O-ribose methylation and pseudouridylation, respectively. As well, many ‘‘orphan snoRNAs’’ lack apparent rRNA or snRNA targets, perhaps suggesting other regulatory targets. Small RNA libraries usually contain a population of reads, sometimes quite substantial in number, from rRNAs, transfer RNAs (tRNAs), and snoRNAs. As routine turnover of these abundant ncRNAs generates shorter species, most miRNA annotators set aside reads matching known ncRNAs. On the other hand, the presence of reads from known ncRNAs in Ago immunoprecipitates (IP) can provide a rationale to consider them further. For example, analysis of human Ago1-IP and Ago2-IP revealed enrichment of duplex reads derived from a hairpin in the ACA45 snoRNA/mir-1839, which were established as Drosha/DGCR8 independent and Dicer dependent (Babiarz et al., 2008; Ender et al., 2008). Similarly, the snoRNA GlsR17 in Giardia lamblia generates a Dicer-dependent functional miRNA (Saraiya and Wang, 2008). These studies prompted reevaluation of other snoRNA-derived (sdRNA) reads, and it is now documented that sdRNAs are frequently recovered from both C/D and H/ACA box classes (Babiarz et al., 2011; Brameier et al., 2011; Ono et al., 2011; Scott et al., 2009; Taft et al., 2009). In some cases, these have been further shown to be dependent on Dicer, to associate with Ago complexes, and to direct detectable repression of complementary targets, generalizing the notion of dual-function snoRNAs that have miRNA activity (Figure 2). Still, there remains good reason to be wary in the functional interpretation of sdRNAs. Although many sdRNAs map with regional preference across snoRNA precursors, this alone is not definitive evidence for a specific biogenesis pathway, as opposed to reflecting more stable degradation fragments. In addition, the simple presence of sdRNAs in Ago-IP libraries may not necessarily reflect genuine residence in Ago, as some abundant cellular species may simply fail to be sufficiently depleted in IP reactions. Nevertheless, there are now clearly many compelling sdRNA substrates that provide a basis for future detailed biochemical analyses of their biogenesis or function. miRNAs from tRNAs Analogous to cases of snoRNA-derived miRNAs, some tRNAderived RNAs (tdRNAs) also contribute to the miRNA pool. One of the first examples came from deep sequencing of mouse embryonic stem cells (mESCs) deleted for dgcr8 or dicer (Babiarz et al., 2008). With the tRNA-Ile/mir-1983 locus, a population of fairly heterogeneous reads was recovered, including reads that spanned its intron or included the untemplated 30 CCA seen in mature tRNAs. However, a species from the 30 end of

Molecular Cell

Review Pol III

Figure 2. Summary of tRNA and snoRNADerived Small RNAs

Pol II tRNA-Ile/ mir-1983

3’ trailer

5’ leader

UU

Alternate folding

pre-tRNA

Box C/D or H/ACA snoRNAs

RNaseP UU

Dicer

CCA

Ago

3’ tRNA trailer

non-AGO associated / other complexes?

mature tRNA

RNaseZ

UU

Degradation

Stress

Ago3/4 tRNA halves

During typical tRNA maturation, the 50 leader and 30 trailer of the pre-tRNA are removed by RNase P and RNase Z cleavages, respectively, followed by 30 CCA addition. Subfragments of tRNAs are frequently observed, many of which reflect routine tRNA turnover and are not regulatory in nature. However, a number of specific pathways have been observed that extend the regulatory range of tRNA loci. In the case of tRNA-Ile/mir-1983, an alternative fold of the pre-tRNA adopts an extensive hairpin that permits cleavage by Dicer. Some mature tRNA cloverleafs may also serve as Dicer substrates and/or generate subfragments that load Ago proteins; this is most prominent in Tetrahymena where the Piwi protein Twi12 carries 30 tRNA fragments exclusively. Some RNaseZgenerated tRNA 30 trailers associate preferentially with Ago3/4 and regulatory activity of these trailers was reported, although this is not necessarily mediated by Ago complexes. Under stress conditions, mature tRNAs are cut into halves, which may associate with unknown complexes to exert regulatory roles. A number of box C/D and box H/ACA snoRNAs can also give rise to Ago-associated, miRNA-like species in a Drosha/DGCR8independent, Dicer-dependent manner.

non-AGO associated / other complexes?

the pre-tRNA (miR-1983) was Dicer dependent but DGCR8 independent, suggesting its identity as a noncanonical miRNA (Babiarz et al., 2008). Interestingly, the tRNA-Ile precursor was predicted to adopt different folds (Figure 2). One formed a typical tRNA cloverleaf, which is cleaved near its 50 end by RNase P and near its 30 end by tRNase Z, prior to CCA addition. However, the terminal sequences normally removed by RNase P/Z can also base pair, thereby extending the duplex base of the tRNA hairpin to present a plausible Dicer substrate. Therefore, alternative conformations can determine entry into different biogenesis pathways. Further study provided additional evidence for other Dicerdependent tdRNAs, tdRNA accumulation in Ago complexes, and/or modulation of tdRNA levels by Ago availability (Cole et al., 2009; Haussecker et al., 2010). As with snoRNAs, caution is warranted in the general interpretation of tRNA fragments that appear in small RNA libraries, and the population of any specific read may actually be contributed through a combination of generic degradation and specific Dicer processing. Moreover, as the Dicer-cleaved product of a tRNA-Gln is 30 modified and inefficiently loaded in Ago complexes (Cole et al., 2009), Dicer processing does not guarantee Ago loading. Still, the collected studies provide ample precedent that some abundant tdRNAs comprise miRNAs. Other tRNA fragments have been cloned, including tRNA halves that accumulate during starvation or oxidative stress (Pederson, 2010). Relatively little is known about their function, but they seem unlikely to be via Argonaute proteins owing to their large size (>35 nt) and their existence in S. cerevisiae, which lacks RNA interference (RNAi)/miRNA pathways altogether. However, the Tetrahymena Piwi protein Twi12 carries smaller, 18–22 nt species that derive nearly exclusively from the 30 ends of mature tRNAs (Couvillion et al., 2010). Their function is

not known, but Twi12 itself is an essential gene. Thus, intersections between tdRNAs and Argonaute pathways deserve further study. tRNaseZ-Derived miRNAs Maturation of canonical miRNAs generates several byproduct species, including flanking miRNA offset reads (moRs) produced by Drosha cleavage and free terminal loops produced by Dicer cleavage (Berezikov et al., 2011; Shi et al., 2009). In analogous fashion, other small RNA species are released during tRNA maturation (Pederson, 2010). For example, tRNase Z cleavage of pre-tRNA transcripts releases
18–25 nt 30 tRNA trailers (Lee et al., 2009). While these may be byproducts, their nonstoichiometric accumulation relative to cognate mature tRNAs may suggest potential functional activity. While this is not necessarily mediated by Ago proteins, a number of 30 tRNA trailers are responsive to Ago levels (Haussecker et al., 2010) (Figure 2). These observations set a precedent that tRNase Z may define the ends of some miRNA species, independently of Drosha. This proved to be the case for miRNAs encoded by murine g-herpesvirus 68 (MHV68). Each of the
20 MHV68 miRNAs maps to tandem hairpins located immediately downstream of a tRNA moiety, suggesting their expression as tRNA-fusions from Pol III promoters (Pfeffer et al., 2005; Reese et al., 2010). In some cases, miRNAs were cloned from both of the tandem hairpins, but in other cases one of the hairpins is preferentially processed into stable small RNAs. These hairpins lack additional ‘‘lower stem’’ pairing indicative of Drosha/DGCR8 binding. Indeed, the production of MHV68 miRNAs is Drosha independent and instead dependent on tRNase Z, which cleaves the 50 ends of the MHV68 pre-miRNA hairpins (Bogerd et al., 2010) (Figure 1B). The mechanism that defines the 30 end of the internal hairpin has not been definitively established, and it could involve an endonuclease that separates the tandem hairpins. However, Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc. 895

Molecular Cell

Review the existence of U-rich stretches at the end of both foldbacks in these tandem arrangements suggests that alternative pol III termination may define the 30 ends. The resulting pre-miRNAs are further processed into mature miRNAs by Dicer (Bogerd et al., 2010). The tRNA-miRNA system is flexible, since the tRNA, miRNA hairpin and the pol III promoter can be readily exchanged (Bogerd et al., 2010). Remarkably, artificial tRNA-short hairpin RNA (shRNA) chimeric expression cassettes were optimized to generate functional siRNAs before elucidation of the MHV68 pathway (Scherer et al., 2007). In these constructs, only a single shRNA hairpin is introduced after the tRNA, and the shRNA ends are thus defined by tRNase Z on the 50 end and by the pol III terminator on the 30 end. The reason for the tRNA-tandem hairpin layout in the MHV68 genome is unclear (Pfeffer et al., 2005; Reese et al., 2010), but its pervasive nature suggests that it has been selected for some functional reason. Endo-shRNAs The first strategies for transgenic RNAi in mammalian cells used pol III-driven shRNAs, for which direct definition of hairpins by transcription permits their processing by Dicer (Medina and Joshi, 1999). Years later, the concept that transcription might determine the ends of some pre-miRNAs was extended to endogenous shRNAs (endo-shRNAs). This is currently a catchall category for DGCR8-independent, Dicer-dependent loci where at least one pre-miRNA hairpin end is generated by transcriptional initiation or termination (Figure 1B). Multiple mechanisms may be involved in their biogenesis, just as with the different flavors of mirtrons. The earlier mentioned tRNA-Ile/mir-1983 is analogous to synthetic shRNAs, in which both pre-miRNA ends are determined by transcription. In the case of mir-320, its 5p species are strongly under-represented relative to 3p reads. While this might be influenced by highly asymmetric loading of miRNA/ star duplexes, 50 RACE detected a processed end corresponding to the 50 end of the hairpin (Babiarz et al., 2008). In principle, if this reflected a genuine transcription start, the resulting 50 triphosphates of 5p reads would be inefficiently ligated by the standard miRNA cloning protocol and thus depleted from libraries. However, a mechanism to determine the 30 end of pre-mir-320 has not yet been elucidated. As well, 50 -tailed endo-shRNAs exist (Babiarz et al., 2008), for which the removal of 50 flanks may potentially be analogous to biogenesis of 50 -tailed mirtrons. siRNAs and miRNAs from Endo-siRNA Pathways In many invertebrates and plants, the RNAi pathway mediates antiviral defense by generating siRNAs from dsRNA aspects of viral life cycles (Ding and Voinnet, 2007). However, nematode and fly mutants that impair exogenous RNAi are virus sensitive, but RNAi are otherwise viable, fertile, and exhibit fairly normal morphology. In mammals, the execution of antiviral defense by the interferon pathway suggested for some time that endogenous RNAi might not even be permissible. However, a broader appreciation of endo-siRNA pathways emerged from deep sequencing studies (Okamura and Lai, 2008). In Drosophila, both somatic and germline tissues are broadly competent to utilize Dcr-2 to cleave endo-siRNAs from transposable elements (TEs), cis-natural antisense transcripts 896 Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.

(cis-NATs) typically comprising convergently transcribed 30 untranslated regions (UTRs), and from long hairpin RNAs (hpRNAs) comprising extensive duplex structure (Chung et al., 2008; Czech et al., 2008; Ghildiyal et al., 2008; Kawamura et al., 2008; Okamura et al., 2008a, 2008b) (Figure 1B). Although endo-siRNAs from all of these substrates are preferentially loaded into AGO2, a subset load into AGO1 and effectively comprise a subpopulation of miRNA. The capacity for endo-siRNA biogenesis in vertebrate cells is more limited than in Drosophila, due to the propensity for dsRNA to activate the interferon response. However, certain cell types, such as mESCs, oocytes, and preimplantation embryos, are tolerant of dsRNA and can use these triggers to mount specific RNAi responses (Paddison et al., 2002; Svoboda et al., 2000; Yang et al., 2001). In yet another example of experimental manipulation preceding the elucidation of underlying endogenous pathways, ESCs and oocytes were later found to express diverse endo-siRNAs from long duplexed precursors (Babiarz et al., 2008; Tam et al., 2008; Watanabe et al., 2008). In addition to the endo-siRNA classes reported in fly, mouse oocytes express abundant siRNAs from dsRNA formed by antisense transcribed pseudogenes hybridized to their sense counterparts (Tam et al., 2008; Watanabe et al., 2008). Interestingly, endosiRNAs from many pseudogene:sense pairs were inferred to be functional, based on broad upregulation of cognate target mRNAs in microarray analysis of dicer/ oocytes (Tam et al., 2008). Although it is not known whether mammalian endosiRNAs are sorted to a specific Ago protein, as in Drosophila, it is presumed that endo-siRNAs resident in mammalian Ago2 mediate the bulk of target regulation via slicing. miR-451 and Dicer-Independent miRNA Biogenesis Initial computational studies of Drosophila canonical miRNAs revealed a characteristic pattern of evolutionary divergence for conserved miRNAs, in that the terminal loop evolves much more quickly than does either miRNA or miRNA* species on the hairpin arms (Lai et al., 2003). Although this was defined on the basis of pairwise alignment of two Drosophila species, it was later found to apply across canonical miRNAs and mirtrons among Drosophilid and vertebrate genomes (Berezikov et al., 2007; Berezikov et al., 2005; Flynt et al., 2010; Okamura et al., 2007). A prominent exception to the rule of preferred loop divergence occurs with vertebrate mir-451. Its terminal loop, like its mature products, is completely conserved across all vertebrates from human to fish. In contrast, its presumed miRNA* species, that is, the hairpin sequence complementary to mature miR-451, contains multiple divergent positions in mir-451 orthologs (Yang et al., 2010). Moreover, its mature cloned species extend over the terminal loop instead of being confined to a hairpin arm, and longer cloned products sharing a 50 end but extending to 30 nt could be recovered in small RNA libraries. All of these properties suggested that miR-451 is not generated by the canonical miRNA pathway. Detailed study of mir-451 homologs from human, mouse, and zebrafish revealed its maturation by an unexpected pathway, the first known to be independent of Dicer (Cheloufi et al., 2010; Cifuentes et al., 2010; Yang et al., 2010). pri-mir-451 is initially cleaved by Drosha/DGCR8 to generate a short pre-miRNA

Molecular Cell

Review Table 1. The Impact of Noncanonical miRNA Pathways and Core Factor Activities on Knockout Phenotypes Phenotypic Comparison

Biological Setting

Relevant Noncanonical Pathways

drosha/dgcr8-KO = dicer-KO

Many places

Bulk phenotypes due to loss of canonical miRNAs

drosha/dgcr8-KO < dicer-KO

ESCs

Roles for Drosha/DGCR8-independent miRNAs (endo-shRNAs, hp-siRNAs?)

drosha/dgcr8-KO < dicer-KO

Brain

Roles for Drosha/DGCR8-independent miRNAs (snoRNAs, mirtrons?)

drosha/dgcr8-KO < dicer-KO

Oocytes

miRNA activity suppressed; endo-siRNAs functional

dicer-KO < drosha-KO

Thymocytes

Possible roles for direct mRNA cleavages by Drosha/DGCR8

drosha/dgcr8/ago2-KO < dicer-KO

Retinal epithelium

Roles for direct Dicer cleavage of Alu dsRNA

dicer-KO < ago2-KO (or Slicer-dead)

Erythropoiesis

Dicer-independent miR-451 and/or other miRNAs?

dAGO1+dAGO2-KO < dgcr8-KO = dicer-1-KO

Fly olfactory neurons

Potential Ago-independent functions of DGCR8/Dicer-1?

with only
18 bp of duplex stem, too short to serve as a Dicer substrate. Instead, pre-mir-451 is loaded directly into Ago proteins (Figure 1C). Those hairpins that enter non-slicing Ago proteins (e.g., Ago1) cannot be matured further, while those that load Ago2 are sliced on their 30 hairpin arm, as guided by the 50 end of the hairpin, yielding a 30 nt Ago-cleaved species. This is subject to a 30 resection activity that trims
7 nt to leave the dominantly cloned 23 nt miR-451; the relevant nuclease(s) remains to be identified. Altogether, the collected studies reveal diverse Drosha-independent and Dicer-independent strategies for miRNA biogenesis, and there is more. For example, the vault noncoding RNA generates a Drosha-independent miRNA (Persson et al., 2009). Curiously, insertion of canonical pri-mir-124 into the Sindbis RNA virus yields functional miR-124 (Shapiro et al., 2010). Sindbis-mir-124 matures cytoplasmically, since miR-124 accumulated in dgcr8 null cells and cells depleted for Exportin-5 (Shapiro et al., 2010). The strategy by which a cellular miRNA matures when inserted into an RNA virus is currently a mystery. Part II: Biological Implications of Alternative miRNA Pathways Use of miRNA Pathway Mutants to Infer miRNA-Dependent Phenotypes A hallmark of genetic analysis is that mutants with similar phenotypes can often be ordered within a common pathway. Although the molecular consequences of lacking the panel of core miRNA pathway components have been extensively characterized, only a few biological settings have been subjected to detailed phenotypic comparison. In some cases, mutants of different core miRNA components do present similar phenotypes. For example, conditional knockout of dicer and dgcr8 during skin development were indistinguishable, causing rough flaky skin, defects in hair follicle downgrowth, ectopic apoptosis, and lethality by 5–6 days after birth (Yi et al., 2009). Similarly, conditional knockout of drosha and dicer within the T cell compartment induced highly overlapping phenotypes, including loss of Foxp3+ cells and lethality due to spontaneous inflammatory diseases by
3 weeks (Chong et al., 2008). Such studies logically support the notion that the major phenotypes of core miRNA biogenesis mutants are attributable to miRNA loss, and certainly the canonical pathway generates the strong majority of miRNA species. Nevertheless, as more studies

are conducted, phenotypic differences among core miRNA pathway members have begun to emerge (Table 1). Interpretation of their differences is limited by the fact that vertebrate studies have focused heavily on dicer mutants, and have rarely been performed in parallel with drosha, dgcr8, and/or ago knockouts. However, given the variety of Drosha/DGCR8-independent pathways, e.g., mirtrons and endo-siRNAs, it is expected that loss of Dicer should exhibit some differences with drosha or dgcr8 mutants. Reciprocally, substrates that are uniquely cleaved by Drosha or by Dicer may cause additional differences. Finally, Ago2 processing of Dicer-independent species may underlie yet other phenotypic distinctions. We discuss here several examples, which collectively suggest that differences among mutants in canonical miRNA machinery may continue to grow as they are scrutinized further. dicer versus dgcr8 Mutants in ESCs and Brain: Microprocessor-Independent Dicer Substrates? Comparison between mESCs conditionally deleted for dgcr8 and dicer revealed general similarities, including strong defects in cell proliferation and differentiation (Kanellopoulou et al., 2005; Murchison et al., 2005; Wang et al., 2007). Rescue experiments using miRNA mimics introduced into mutant cells attributed some phenotypes to specific canonical miRNAs, such as control of G1-S transition by members of the mir-290 cluster that target cell-cycle factors (Wang et al., 2008). On the other hand, dicer knockout cells exhibited noticeably stronger defects than dgcr8 knockouts. For example, compared to dgcr8 knockout cells, dicer knockout cells are much more difficult to grow out after Cre-mediated excision, and loss of dicer causes a more complete block in directed differentiation assays (Kanellopoulou et al., 2005; Murchison et al., 2005; Wang et al., 2007). While the mechanistic basis for these differences remain to be understood, there exists a population of DGCR8-independent mirtrons, endo-shRNAs, and hp-siRNAs in ESCs (Babiarz et al., 2008). The rescue approach, searching for small RNA mimics that preferentially improve the ability to differentiate dicer/ cells, relative to dgcr8/ cells, may prove informative in uncovering biological activities of noncanonical ES miRNAs. In particular, the relatively high abundance of the endo-shRNAs mir-320 and mir-484 and the hp-siRNA short interspersed repetitive element (SINE) locus suggests them as candidates for functional study. Comparison of dicer and dgcr8 conditional knockout in postmitotic neurons revealed further phenotypic differences. Both Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc. 897

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Review genotypes caused microcephaly and lethality, but deletion of dicer resulted in earlier lethality and more severe morphological abnormalities (Babiarz et al., 2011). Small RNA analysis revealed diverse DGCR8-independent, Dicer-dependent small RNAs in brain, of which a majority (by total abundance) derived from snoRNAs, followed by mirtrons. By comparison, these classes comprised only a small minority of noncanonical miRNAs in ESCs (Babiarz et al., 2008). Therefore, cell-specific deployment of different classes of noncanonical miRNAs may underlie distinct underpinnings to phenotypic differences between dicer and dgcr8 conditional knockouts in different settings. dicer versus dgcr8 Mutants in Oocytes: Suppression of miRNA-Mediated Regulation? The differences between loss of DGCR8 and Dicer are unexpectedly more profound in oocytes. dicer knockout oocytes exhibit strong phenotypes, including defects in meiotic spindle assembly and chromosome condensation, inability to complete even the first cell division, and broad alterations across the transcriptome (Murchison et al., 2007; Tang et al., 2007). While this might plausibly reflect early and broad roles for maternally inherited miRNAs, attempts to identify a signature of miRNA target site enrichment in transcripts upregulated in dicer mutant oocytes notably did not succeed. Instead, evidence was obtained that TEs, especially mouse transposon (MTs) and SINEs, were deregulated in this condition (Murchison et al., 2007). These observations helped motivate the search for endo-siRNAs in oocytes, which as mentioned are generated by select TEs and complementary gene:pseudogene pairs (Tam et al., 2008; Watanabe et al., 2008). Curiously, the mRNA targets that are functionally repressed by endo-siRNAs are enriched for genes involved in microtubule dynamics, suggesting a possible connection to the spindle defects of dicer mutant oocytes. A surprise came with the analysis of dgcr8 mutant oocytes. While these exhibited the same strong and complete loss of canonical miRNAs as dicer mutant oocytes, the loss of maternal DGCR8 was compatible with normal dynamics and morphology of oocyte maturation, and paternally rescued embryos were subsequently viable and fertile (Suh et al., 2010). Although zygotic DGCR8 is obviously required for embryonic development, due to general roles for miRNAs, maternal-zygotic dgcr8 mutants developed normally until the blastocyst stage. Absent morphological defects, a molecular signature of dgcr8 loss might still have been detectable. However, the transcriptomes of wildtype and dgcr8/ oocytes were essentially identical, with a scant three transcripts differing significantly (one being the floxed dgcr8 transcript itself). This suggested that miRNAs are dispensable for oocyte maturation and early mammalian development. Microarray analysis showed that known mirtron and endo-shRNA seeds were not enriched among deregulated transcripts in dicer/ oocytes, pointing to endo-siRNAs as likely to be the key small RNA regulators in this setting. Explicit tests of regulatory activity of miRNAs during oocyte maturation provided another surprise. The accumulation of mature miRNAs is dynamic during this process (Tang et al., 2007), and their function was reflected by the repression of reporters bearing perfectly matched sites to abundant miRNAs, presumably by slicing. However, their capacity to repress bulged sensors was progressively lost during oocyte maturation, as was 898 Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.

the localization of miRNA-targeted transcripts to P bodies (Ma et al., 2010). Understanding how general miRNA effector activity might be antagonized is a challenge for the future, but the retention of slicing in oocytes implies a selective defect in Argonauteassociated effectors. One wonders whether aberrant activity of such a miRNA-suppressing pathway in later development may underlie disease or cancer. mRNA Cleavage by Drosha: Direct Regulation of dgcr8, and of Other mRNAs? A current mystery in miRNA biogenesis regards how Drosha/ DGCR8 complex specifically recognizes pri-miRNA substrates. The main determinant reported is that DGCR8 recognizes the junction between the single-stranded and double-stranded region of the pri-miRNA hairpin base (Han et al., 2006). However, as presumably millions of transcript structures juxtapose singlestranded and double-stranded regions, it is unclear how primiRNAs are selectively identified. Perhaps Drosha is not entirely selective for miRNAs. Bacterial RNase III enzymes mature ribosomal RNA, and yeast RNase III not only shares this activity but also matures certain snRNAs and snoRNAs (Drider and Condon, 2004). Consistent with this, mammalian Drosha was reported to be involved in ribosomal RNA biogenesis (Fukuda et al., 2007; Wu et al., 2000). Note, though, that defective rRNA processing was not observed after drosha knockout in the lymphoid system (Chong et al., 2008). As the depth of sequencing catalogs increases, small RNA reads mapped to mRNA hairpins have begun to emerge (Berezikov et al., 2011). The best-characterized mRNA target of Drosha happens to encode its cofactor, DGCR8. Here, hairpins within its 50 UTR and coding region are cleaved by Drosha, yielding a low level of small RNAs. However, bulk dgcr8 ‘‘pre-miRNA’’ hairpins are not destined for miRNA production, since cell fractionation showed them to be nuclearly restricted (Han et al., 2009). Instead, the main function of dgcr8 cleavage is to repress accumulation of DGCR8 protein (Figure 3A). Reciprocally, Drosha protein is unstable in the absence of DGCR8. Together, cross-regulation tunes their respective levels for appropriate heterodimer function (Han et al., 2009; Triboulet et al., 2009). Cleavage of pasha 50 UTR hairpins by Drosha is conserved in D. melanogaster (Han et al., 2009; Kadener et al., 2009), indicating that it is an ancient regulatory strategy. With this precedent, one may wonder whether Drosha has a more general role in mRNA cleavage. One study concluded that dgcr8 is a fairly specific Drosha mRNA target (Shenoy and Blelloch, 2009). However, another study reported certain stronger phenotypes upon conditional knockout of drosha during T cell development, compared to dicer (Chong et al., 2010). Amongst transcripts uniquely upregulated in DN3 cells upon drosha knockout, a number bore potentially structured regions that generated
20–25 nt small RNA reads and could be cleaved by Drosha in vitro (Chong et al., 2010). At present, such mRNA cleavage has not been causally linked to phenotypes. Moreover, observed changes in gene expression may have arisen from differential representation of cell types, since substantial canonical miRNAs were still sequenced in the DN3 knockouts of drosha and dicer (Chong et al., 2010). Still, ‘‘degradome’’ sequencing of mRNA fragments bearing 50 phosphates revealed Drosha-dependent, Ago2-independent cleaved

Molecular Cell

Review A

B

Pol II

Pol III

Drosha 5’ AAAA...3’ mRNA

DGCR8

Alu dsRNA transcripts

Dicer 5’

cytotoxicity

AAAA...3’

mRNA degradation

inert cleaved Alu fragments

Figure 3. Direct Cleavage of RNA Substrates by RNase III Enzymes RNase III enzymes may have regulatory roles that are independent of miRNA production. (A) mRNAs bearing short hairpin structures in untranslated or coding regions can be recognized and cleaved by Drosha/DGCR8, resulting in mRNA destabilization. The best-characterized example of this is cleavage of the dgcr8 mRNA by Drosha. (B) The pol III-transcribed long double-stranded Alu transcripts are toxic to cells unless they are cleaved by Dicer to short, inert fragments. In addition, direct dicing of viral replication intermediates may contribute to antiviral defense in Drosophila.

mRNAs that may include direct Drosha targets (Karginov et al., 2010). Finally with Kaposi’s Sarcoma-associated Herpesvirus, Drosha cleavage of miRNAs located in the 30 UTR of the viral transcript KapB downregulates this mRNA in cis, independent of activity of the miRNAs as trans regulators (Lin and Sullivan, 2011). It will be a challenge for the future to clarify the extent to which mRNA cleavage by Drosha influences gene expression and, if so, how these targets are selected appropriately. dicer-Specific Function in Macular Degeneration: Direct Dicing of Alu RNAs? If Drosha can regulate messages by direct cleavage, one may wonder whether Dicer might do the same. A recent study of human patients with geographic atrophy (GA), an age-related macular degeneration disease of the retinal pigmented epithelium (RPE), showed reduction of dicer mRNA and protein but little change in other core miRNA pathway components (Kaneko et al., 2011). This led them to systematically analyze conditional knockouts for most of the major miRNA pathway members (drosha, dgcr8, dicer, ago2, ago1, ago3, ago4, and tarbp2). Impressively, only ablation of dicer recapitulated the GA phenotype, suggesting that this disease is not due to general alteration in miRNA activity. Using an antibody that recognizes long dsRNA, the authors found that dsRNA accumulates in human GA eyes, as well as in human RPE cells and mouse eyes depleted of Dicer. The dsRNA population was cloned and found to include Alu repeat RNAs of
300 nt. Their accumulation was a specific property

of Dicer-depleted cells and not other genotypes, and loss of Dicer deregulated Alu but not other retrotransposon transcripts. In fact, functional tests demonstrated that accumulation of Alu RNAs is cytotoxic and that injection of in vitro-transcribed Alu induced GA. On the other hand, injection of Dicer-cleaved Alu small RNAs, or other noncoding RNAs, had no effect. Together, these tests supported a model in which Dicer exerts a miRNA-independent function in cleaving Alu dsRNAs to render them inert (Kaneko et al., 2011) (Figure 3B). Although no single Ago gene (including Ago2-Slicer) is needed to prevent GA, it remains to be seen whether cleaved Alu siRNAs also function as Ago-loaded species. However, the notion of direct dicing as a biological function bears similarity to studies of persistent viral infection of Drosophila cultured cells. It is well established that a Dicer-2/AGO2-mediated siRNA pathway executes antiviral defense in flies (Wang et al., 2006). However, bulk viral siRNAs generated by Dicer-2 in latently infected cells appear to associate poorly with effector complexes. Those that are successfully loaded enter AGO2 (Czech et al., 2008), but bulk viral siRNAs did not associate with either AGO2 or AGO1 (Flynt et al., 2009). One interpretation of these findings is that direct dicing of the viral replication intermediate plays a substantial role in controlling persistent viral infection of Drosophila cells. Can the theme of noncanonical substrates of core miRNA factors be extended beyond Drosha and Dicer? Mammalian Exp-5 was recently reported to directly transport dicer mRNA to the cytoplasm (Bennasser et al., 2011). Given the topology of preferred Exp-5 binding to hairpins with 30 overhangs, it is not clear how Exp-5 binds dicer transcripts. Nevertheless, this example suggests it may be worth considering whether Exp-5 transports other non-miRNA substrates. ago2 versus dicer Mutants in Hematopoietic System: Slicer-Mediated Functions? The elucidation of miR-451 biogenesis raises a new wrinkle, in that dicer knockout cells do not universally remove all miRNAs. Phenotypes of knockin mice bearing the ago2 Slicer-deficient allele are provocative, including full perinatal lethality and prominent anemia (Cheloufi et al., 2010). While miR-451 is clearly deficient in this genetic condition, the loss of miR-451 per se does not explain the Ago2-Slicer mutant phenotype. Instead, mir-451 deletion mutants are fully viable and exhibit only mild anemia, although this presents a more substantial problem upon oxidative challenge (Patrick et al., 2010; Rasmussen et al., 2010; Yu et al., 2010). This may be taken to support the existence of essential miRNA-directed cleavage events. Certainly there are a number of documented endogenous mRNA cleavages programmed by miRNAs, although none are known to be required for hematopoiesis or viability (Karginov et al., 2010; Shin et al., 2010; Yekta et al., 2004). Additional scenarios for the requirement of Ago2 Slicer activity include that it may process other Dicer-independent miRNAs yet to be identified, is required more generally for miRNA biogenesis (Diederichs and Haber, 2007), or potentially regulates targets independently of mature miRNA guides. Consistent with the latter possibility, Ago2 is capable of using guides larger than mature siRNAs/miRNAs to direct target cleavage (Tan et al., 2009). Very recently, the direct association of Ago2 with murine Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc. 899

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Review transcripts was studied genomewide, and compared between normal and dicer/ ESCs (Leung et al., 2011). As expected, a dominant Dicer-dependent signature of target sites complementary to the seeds of highly expressed ES miRNAs was seen. Less expected, though, was the observation of Dicer-independent Ago2 targeting signatures, which included G-rich motifs. Although the mechanistic significance of this remains to be explored further, evidence was presented that these motifs correlate with preferential conservation and target derepression in ago2/ ESCs. These data may support the notion that Ago2 is targeted to certain transcripts independently of mature miRNAs, perhaps reflecting intrinsic RNA affinity, or perhaps guided by RNAs independent of Dicer. Most profilings of Argonaute-associated RNAs have focused exclusively on <30 nt species and mRNAs. While miRNA-sized species are the predominant contents of Ago proteins in the short RNA fraction, this could be biased by the fact that their size is very stereotyped. In principle, larger heterogenously-sized cargoes might not appear to be distinct from background in total RNA labelings. Therefore, it may be informative to broaden sequencing surveys to search for intermediate-sized RNAs that might associate with Ago proteins. Drosophila ago1/ago2 Mutants versus pasha/dcr-1 Mutants in the Olfactory System To date, most efforts to infer miRNA functions from miRNA pathway components have focused on the biogenesis factors, but certainly analysis of Ago mutants should be informative. For example, Drosophila ago1/ago2 double-mutant embryos exhibited phenotypes more severe than either single mutant (Meyer et al., 2006). There are four mammalian Argonautes, but their genetic analysis is aided by the close linkage of ago1/3/4, which can be deleted in a single event. Such tripleknockout ESCs maintain normal levels of miRNA-mediated silencing, indicating that Ago2 is capable of supporting siRNA and miRNA activity (Su et al., 2009). Deletion of ago2 in this background yielded quadruple ago1–4 knockout cells that exhibit strong growth defects (Su et al., 2009), although such mutant cells remain to be examined in intact mice. In principle, as removal of the effector proteins should effectively abolish all mi/siRNAs, regardless of their biogenesis history, one might intuit that this situation should be ‘‘worse’’ than any individual biogenesis factor. However, a few years ago, a genetic screen in Drosophila olfactory projection neurons revealed two mutants exhibiting a distinct set of dendritic and axonal mistargeting phenotypes (Berdnik et al., 2008). These mutants disrupted dcr-1 and pasha (dgcr8), implying a common function of the miRNA pathway in controlling olfactory wiring. Surprisingly, neither ago1[k08121] nor ago2[414], as single or double mutants, recapitulated the olfactory system defect. It is possible that this ago1 mutant is not null; however, the insertion alleles isolated for dcr-1 and pasha were not necessarily null either. Moreover, ago1[k08121] is known to have strongly decreased mRNA and protein levels (Kataoka et al., 2001; Okamura et al., 2004). Whether this implies that a very small amount of Ago effector complex suffices for the morphogenesis of projection neurons, or whether there are Ago-independent activities of the miRNA pathway, remains to be clarified. 900 Molecular Cell 43, September 16, 2011 ª2011 Elsevier Inc.

Does Loss of Core miRNA Machinery Reflect the Cumulative Phenotypes of Individual miRNA Mutants? These many examples provide substantial evidence that no single core miRNA pathway component is essential to generate all animal miRNAs. However, a final consideration regards the very expectation that core biogenesis mutants should reflect the cumulative phenotypes of all individual miRNA mutants. In Drosophila, induction of dcr-1 and pasha null clones during wing development results in blistering of the adult wing, but otherwise the integrity of the wing margin remains fully intact (Bejarano et al., 2010). This might be taken as evidence that miRNAs are not required to specify the wing margin, were it not for the fact that deletion of a single miRNA—mir-9a—alone confers fully penetrant wing notching (Li et al., 2006). A trivial explanation might be that residual proteins or miRNAs in mutant clones suffice for normal wing development. However, in vivo sensor assays showed that miR-9a function was lost to a similar extent in dcr-1, pasha, and mir-9a clones (Bejarano et al., 2010). Moreover, similar mutant clones of mir-9a still yielded wing notching. Perhaps most compelling were observations using a 30 UTR sensor for dLMO, a key miR-9a target during wing development (Bejarano et al., 2010; Biryukova et al., 2009): the dLMO sensor was derepressed in dcr-1, pasha and mir-9a clones, but endogenous dLMO protein was derepressed only in mir-9a clones (Bejarano et al., 2010). The simplest interpretation is that there exist other miRNAs whose activity is antagonistic to miR-9a during wing development. More generally, as a majority of animal transcripts may be targeted by miRNAs, and many processes are typically under positive and negative control, it may not be so unexpected for the loss of miRNA biogenesis factors not to phenocopy the loss of specific miRNAs. For example, both positive and negative regulators of peripheral neurogenesis contain target sites for the same miRNAs (Lai et al., 1998; Lai and Posakony, 1997), perhaps explaining why phenotypes associated with loss of miRNA binding sites from individual neural regulators are not recapitulated by dcr-1 clones. In fact during early zebrafish development, both positive and negative regulators of Nodal signaling are repressed by miR-430, such that the inhibition of target sites from individual transcripts is more severe than loss of miR-430 or Dicer (Choi et al., 2007). Therefore, the absence of phenotypes in core miRNA pathway mutants cannot reliably be taken to mean the absence of compelling miRNA functions in a given setting. Conclusion and Outlook The evolutionary flexibility of small RNA pathways is clearly evident from the diversity of animal miRNA and siRNA pathways, and further illustrated by recent studies in fungal systems. For example, certain budding yeasts encode a clear Argonaute ortholog but lack a recognizable Dicer. Detailed investigation of S. castellii revealed that an orphan RNase III enzyme executes dicing, even though this protein has only one RNase III domain instead of the two seen in canonical Dicers, and entirely lacks the usual helicase and PAZ domains (Drinnenberg et al., 2009). IP cloning from the Neurospora crassa Argonaute QDE-2 revealed a diversity of miRNA-like species (Lee et al., 2010), at least one of which is Dicer independent but instead requires

Molecular Cell

Review the RNase III enzyme MRPL3. More surprisingly, some siRNA loci in Neurospora require neither Dicer nor MRPL3, implying yet another nuclease in their biogenesis. Finally, the study of Ago1 complexes from S. pombe dcr1 mutants revealed a system of Dicer-independent ‘‘primal RNAs’’ that prime the RNAi machinery (Halic and Moazed, 2010). Going even further ‘‘out of the box,’’ cell death in C. elegans was found to involve a caspase-dependent cleavage of Dicer, converting it from an RNase into a DNase that fragments chromosomes (Nakagawa et al., 2010). This may cause one to wonder whether other miRNA/RNAi factors may have DNAdirected functions. For example, some archaeabacterial Argonaute proteins preferentially utilize a DNA guide strand (Yuan et al., 2005). Altogether, the collected studies suggest that small RNA researchers have not yet fully appreciated the inventiveness of Nature in defining noncanonical functions of small RNA-associated proteins, which can be incorporated into unexpected pathways and mediate unexpected biology. ACKNOWLEDGMENTS We apologize to authors whose work was not cited due to length restrictions. We thank Katsutomo Okamura and Robert Blelloch for helpful discussion and members of the Lai laboratory for work that inspired this perspective. Work in E.C.L.’s group was supported by the Burroughs Wellcome Fund, the Alfred Bressler Scholars Fund, the Starr Cancer Consortium (I3-A139), and the National Institutes of Health (R01-GM083300). REFERENCES Axtell, M.J., Westholm, J.O., and Lai, E.C. (2011). Vive la diffe´rence: biogenesis and evolution of microRNAs in plants and animals. Genome Biol. 12, 221. Babiarz, J.E., Ruby, J.G., Wang, Y., Bartel, D.P., and Blelloch, R. (2008). Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev. 22, 2773– 2785. Babiarz, J.E., Hsu, R., Melton, C., Thomas, M., Ullian, E.M., and Blelloch, R. (2011). A role for noncanonical microRNAs in the mammalian brain revealed by phenotypic differences in Dgcr8 versus Dicer1 knockouts and small RNA sequencing. RNA 17, 1489–1501. Bartel, D.P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233. Bejarano, F., Smibert, P., and Lai, E.C. (2010). miR-9a prevents apoptosis during wing development by repressing Drosophila LIM-only. Dev. Biol. 338, 63–73. Bennasser, Y., Chable-Bessia, C., Triboulet, R., Gibbings, D., Gwizdek, C., Dargemont, C., Kremer, E.J., Voinnet, O., and Benkirane, M. (2011). Competition for XPO5 binding between Dicer mRNA, pre-miRNA and viral RNA regulates human Dicer levels. Nat. Struct. Mol. Biol. 18, 323–327. Berdnik, D., Fan, A.P., Potter, C.J., and Luo, L. (2008). MicroRNA processing pathway regulates olfactory neuron morphogenesis. Curr. Biol. 18, 1754– 1759. Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E., Plasterk, R.H., and Cuppen, E. (2005). Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120, 21–24. Berezikov, E., Chung, W.-J., Willis, J., Cuppen, E., and Lai, E.C. (2007). Mammalian mirtron genes. Mol. Cell 28, 328–336. Berezikov, E., Robine, N., Samsonova, A., Westholm, J.O., Naqvi, A., Hung, J.H., Okamura, K., Dai, Q., Bortolamiol-Becet, D., Martin, R., et al. (2011). Deep annotation of Drosophila melanogaster microRNAs yields insights into their processing, modification, and emergence. Genome Res. 21, 203–215.

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